It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium. These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes. This may be relevant to the etiology of diarrhea caused by EPEC infection.